CN101407773B - Pseudomonas putida GNA5 bacterial strain and method for preparing D-glucosaminicacid using the same - Google Patents
Pseudomonas putida GNA5 bacterial strain and method for preparing D-glucosaminicacid using the same Download PDFInfo
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- CN101407773B CN101407773B CN2008101562265A CN200810156226A CN101407773B CN 101407773 B CN101407773 B CN 101407773B CN 2008101562265 A CN2008101562265 A CN 2008101562265A CN 200810156226 A CN200810156226 A CN 200810156226A CN 101407773 B CN101407773 B CN 101407773B
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a Pseudomonad putida GNA5 strain and a D-glucosamine acid preparation method, and can transform D-glucosamine to D-glucosamine acid in high efficiency by utilizing the Pseudomonad putida GNA5 strains (CCTCCM208138) that are filtered and preserved and adopting an oxidative fermenting or cell resting transformation method. The D-glucosamine acid preparation method solves the problems that the strain cannot endure high-concentration D-glucosamine and has strong metabolic capability of the product D-glucosamine acid in a microorganism transforming preparation process of D-glucosamine to D-glucosamine acid.
Description
Technical field
The invention belongs to the using microbe field, be specifically related to a kind of pseudomonas putida (Pseudomonasputida) GNA5 bacterial strain and prepare the method for D-glucosamine acid.
Background technology
D-glucosamine acid (D-Glucosaminic acid), chemistry 2-amino-3,4,5 by name, 6-tetrahydroxy caproic acid is a kind of very promising food flavouring, can be used as the synthesis material of chiral amino acid derivative with antiviral, antitumous effect etc.This material good biocompatibility, the title complex with metal chromium ions forms has medical blood sugar reducing function; The title complex that forms with metal platinum is having application promise in clinical practice aspect the treatment cancer.In addition, D-glucosamine acid is expected to be used widely in the chromatographic separation of charged biomolecules as a kind of positively charged ion coordination agent.
The preparation method of D-glucosamine acid has three kinds in the existing report: chemical oxidization method, Pringsheim (Chem.Ges., 48:680-682.) etc. reported that employing mercuric acetate oxidation D-glucosamine (D-glucosamine) prepares, but this method degree of oxidation is wayward, poor selectivity, yield is low; Enzyme transforming process, (Carbohydr.Res. such as Pezzotti, 340:139-141) utilize glucose oxidase coupling connection catalase, with the D-glucosamine is the acid of feedstock production D-glucosamine, but glucose oxidase has only 2% of glucose to the catalytic efficiency of D-glucosamine, this research and utilization rolls up the consumption of enzyme or prolongs the reaction times and can effectively improve catalytic efficiency, and through enzyme reaction in 72 hours, yield reached 76%; Microbial method prepares D-glucosamine acid and receives publicity because of transformation efficiency is higher, but fermentation middle and high concentration substrate D-glucosamine has certain inhibition to bacterial strain and relevant saccharase, and the concentration of substrate of fermentation oxidation style is no more than 10g/L (Jap.Pat:S59-014787.1984-01-25 in the existing report; Appl.Microbiol.Biotechnol., 66:253-258).Secondly, in general, the bacterial strain that produces D-glucosamine acid has further catastatic ability, often needs to add the accumulation (Jap.Pat:S59-014787.1984-01-25) that inhibitor such as alpha-brominated Sodium Propionate promotes product.Moonmangmee etc. utilize the dry back Gluconobacter frateurii IFO3246 thalline of high density, have overcome phenomenons such as substrate inhibition effectively; In the transformation system, the concentration of substrate D-glucosamine hydrochloride is 100g/L, and the addition of Gluconobacter frateurii IFO 3246 is 0.1g dry mycelium/g substrate, and molar yield reaches more than 80%.Because the thalline addition is big, has brought side reaction, makes transformation efficiency not high.
Summary of the invention
The purpose of this invention is to provide a kind of pseudomonas putida (Pseudomonas putida) GNA5 bacterial strain and prepare the method for D-glucosamine acid with it, solve the oxidative fermentation D-glucosamine and prepare in the D-glucosamine acid process, bacterial strain can not tolerate the high density D-glucosamine and to problem that product D-the glucosaminicacid metabolic capacity is strong.
The present invention is that the sole carbon source and the energy screen the soil sample of different sources with the D-glucosamine hydrochloride, select altogether some strains can the tachymetabolism D-glucosamine bacterial strain, can transform the bacterial strain that D-glucosamine prepares D-glucosamine acid from wherein screening again.Adopt Paper Chromatography to detect the generation situation of D-glucosamine acid in the different incubation time fermented liquids therebetween, choose the high strain Pseudomonas putida GNA5 of transformation efficiency.This bacterial strain has sent Chinese typical culture collection center preservation, and deposit number is preserving number: CCTCCNO:M208138, and preservation date is on September 27th, 2008.
The Pseudomonas putida GNA5 bacterial strain that the present invention screens is a Gram-negative bacteria, and no gemma is shaft-like.Identify through BIOLOG automatic bacterial assessing instrument, show that this bacterial strain and pseudomonas putida (Pseudomonas putida) similarity (sim) is 0.82, show that through 16S rDNA sequential analysis this sequence and bacterial strain pseudomonas putida (Pseudomonas putida) M G-Y2 homology are up to 99%.Therefore, this bacterial strain is pseudomonas putida (Pseudomonas putida) GNA5.
Up to now, there is not bibliographical information pseudomonas putida (Pseudomonas putida) can accumulate D-glucosamine acid
The present invention is under the situation of not adding any inhibitor, and with the strain Pseudomonas putida GNA5 that screening obtains, oxidative fermentation directly accumulates D-glucosamine acid in containing the substratum of D-glucosamine.
Described D-glucosamine can the D-glucosamine hydrochloride or the form of D-glucosamine vitriol add.
D-glucosamine hydrochloride or D-glucosamine sulfuric acid hydrochlorate can be mixed with the aqueous solution earlier, regulate pH to 6.5-7.5 with NaOH again, are added into substratum then.
Content≤the 25g/L of carbon nitrogen source in the described substratum, carbon source preferred carbohydrate, especially glucose wherein, addition≤10g/L; Nitrogenous source wherein is inorganic and organonitrogen, inorganic nitrogen-sourced ammonium chloride or ammonium nitrate, the content≤5g/L of can be; Organic nitrogen source can be yeast powder, urea, peptone or extractum carnis, addition≤10g/L.
When being added into D-glucosamine hydrochloride in the substratum or D-glucosamine sulfate concentration, can obtain higher transformation efficiency smaller or equal to 0.4mol/L.
Also comprise following composition in the described substratum: buffer reagent potassium primary phosphate, content≤5g/L; PH regulator agent calcium carbonate content is 5-30g/L; The micro-magnesium salts of somatomedin for example can be selected bitter salt for use; The initial pH of fermented liquid is 4.5-8.5.
Bacterial classification inoculation amount (volume percent) is generally 1%-10%, under 100-200 rev/min of rotating speed stirs, maintains the temperature at 25-40 ℃, and fermentation time was generally 40-80 hour.
The invention provides the method that another prepares D-glucosamine acid, promptly adopt the conversion of resting cells D-glucosamine of Pseudomonasputida GNA5 bacterial strain to prepare D-glucosamine acid, comprise: (1) is carried out routine with Pseudomonas putida GNA5 bacterial strain and is cultivated, fermentation obtains resting cell; (2) D-glucosamine being mixed with solution is biological conversion of substrate; (3) with (1) resting cell, the substrate solution that adds (2) carries out conversion reaction, generates D-glucosamine acid.
In the Pseudomonas putida GNA5 strain fermentation substratum with glucose as carbon source, preferred initial stage stationary phase of fermentation time.With the resting cell that obtains, with cryogenic physiological saline flush away surface residue.D-glucosamine in the reaction can the D-glucosamine hydrochloride or the D-glucosamine sulphate form add; Concentration of substrate is smaller or equal to 0.7mol/L, add-on 0.02-0.2g/mM substrate of resting cell, and the conversion reaction temperature is 25-40 ℃, the conversion reaction time is 1-48 hour.
In sum, the present invention has screened Pseudomonas putida GNA5 bacterial strain, this bacterial strain can tolerate the high density D-glucosamine and to product D-glucosaminicacid metabolic capacity extremely a little less than, can be equipped with D-glucosamine acid by oxidative fermentation method and conversion of resting cells legal system, the transformation efficiency height is good production bacterial strain.
Description of drawings
Fig. 1 is the paper tomographic map of isolated strains fermented liquid.Wherein 1:50mg/L D-glucosamine and 5mg/LD-glucosaminicacid standard substance; 2: substratum; 3: the fermented liquid supernatant of strain Pseudomonas putida GNA5; 4: the fermented liquid supernatant of bacterial strain GNA7.
Specific embodiment
Embodiment 1
The screening procedure of the natural production bacterial strain of this description of test high yield D-glucosamine acid.
Screening and culturing based formulas (g/L) is: D-glucosamine hydrochloride 10, CaCO
33, potassium primary phosphate 1, bitter salt 0.5, SODIUMNITRATE 1, yeast powder 0.5, pH7.0.The soil sample of different sources of taking a morsel joins in the screening culture medium, at 30 ℃, and 120 rev/mins of following shaking culture, liquid amount 10ml/ test tube (25*200mm) was cultivated 5～7 days, replenished vaporization losses moisture with sterile distilled water therebetween; Get enrichment culture liquid 0.1ml, switching is gone in the fresh aseptic screening culture medium, 3 generations of switching enrichment culture, and adopt Paper Chromatography to detect the consumption of D-glucosamine in the enrichment culture liquid and the generation of D-glucosamine acid.
Plate culture medium prescription (g/L) is: glucose 10, potassium primary phosphate 2, NH
4NO
32, bitter salt 0.5, yeast powder 5, agar 18, pH7.0.The dilution of enrichment culture liquid is coated on the plate culture medium, cultivated 1～2 day for 30 ℃; The bacterium colony of selecting well-grown, different shape carries out setting-out to be separated, and obtains single bacterial strain.Single bacterial strain inserted respectively in the fresh aseptic screening culture medium carry out multiple sieve, measure the content of D-glucosamine and D-glucosamine acid in the fermented liquid therebetween, select the fast or stronger bacterial strain of accumulation D-glucosamine acid ability of D-glucosamine consumption.
From accompanying drawing 1, can see, there is D-glucosamine acid to exist in the oxidative fermentation nutrient solution of bacterial strain GNA5, and the spot of D-glucosamine is less lighter, illustrate that bacterial strain GNA5 can transform D-glucosamine and prepare D-glucosamine acid, and has higher conversion capability, so far, screen aimed strain.Adopt BIOLOG automatic bacterial assessing instrument to identify and 16S rDNA sequential analysis, show that this bacterial strain is Pseudomonas putida, called after Pseudomonas putida GNA5.
In the above-mentioned screening process, D-glucosamine in the nutrient solution and D-glucosamine acid adopt Paper Chromatography to detect, and method is as follows: developping agent is an ethanol: Glacial acetic acid: water=6:1:1 (volume ratio); Get the fermented liquid 2 μ l of enrichment culture liquid or pure bacterial strain, use the microsyringe point sample on filter paper; Ply of paper dries up filter paper after analysing end, sprays 0.2% ninhydrin solution and heats colour developing in 5 minutes at 95 ℃.The spot of substrate D-glucosamine and product D-glucosaminicacid all is red-purple, and Rf value (Rf) is respectively 0.65 and 0.21.
Embodiment 2
This description of test utilizes Pseudomonas putida GNA5 bacterial strain oxidative fermentation to prepare the method for D-glucosamine acid.
(1) Pseudomonas putida GNA5 bacterial strain oxidative fermentation in culture medium A prepares D-glucosamine acid.
The A preparation of substratum:
1. D-glucosamine hydrochloride or D-glucosamine vitriol are mixed with the strong solution that concentration is 0.9mol/L, adopt sterilization by filtration degerming (selecting 0.22 μ m film for use).
2. prepare the rest part (g/L) of substratum: glucose 5, potassium primary phosphate 1, bitter salt 0.5, lime carbonate 20, urea 3, yeast powder 2, initial pH=6.5.Afterwards, 121 ℃ of sterilizations 15 minutes.
3. step 1 and 2 gained solution are mixed under gnotobasis, the final concentration that makes D-glucosamine hydrochloride or D-glucosamine vitriol is 0.35mol/L.
Pseudomonas putida GNA5 bacterial strain oxidative fermentation in culture medium A, its inoculum size are 4% (percent by volume), and culture temperature is 30 ℃, 180 rev/mins of shaking speed.Fermenting can prepare the 34.1g/LD-glucosaminicacid in 70 hours, and molar yield is 49.87%.
The D-glucosamine hydrochloride or the D-glucosamine vitriol strong solution of step 1 also can be regulated pH to 6.5-7.5 with NaOH in the culture medium A preparation, and then filtration sterilization.
(2) Pseudomonas putida GNA5 bacterial strain oxidative fermentation in substratum B.
The preparation of substratum B:
1. D-glucosamine hydrochloride/D-glucosamine vitriol is mixed with the strong solution that concentration is 0.9mol/L, adopts sterilization by filtration degerming (selecting 0.22 μ m film for use).
2. prepare the rest part (g/L) of substratum: glucose 5, urea 5, potassium primary phosphate 2, bitter salt 0.5, lime carbonate 10, pH=7.0.Afterwards, 121 ℃ of sterilizations 15 minutes.
3. step 1 and 2 gained solution are mixed under gnotobasis, the final concentration that makes D-glucosamine hydrochloride/D-glucosamine vitriol is 0.14mol/L.
Pseudomonas putida GNA5 bacterial strain oxidative fermentation in substratum B, its inoculum size is 6% (percent by volume), culture temperature is 30 ℃, 180 rev/mins of shaking speed.Fermenting can prepare the acid of 25g/L D-glucosamine in 48 hours, and molar yield is 91.33%.
The D-glucosamine hydrochloride or the D-glucosamine vitriol strong solution of step 1 also can be regulated pH to 6.5-7.5 with NaOH in the substratum B preparation, and then filtration sterilization.
(3) detection method of D-glucosamine and D-glucosamine acid
In this experiment, high performance liquid chromatography (HPLC) detection by quantitative is adopted in D-glucosamine and D-glucosamine acid.Fermented liquid behind the centrifugal 15min, obtains supernatant liquor at 6000 rev/mins under 4 ℃ of conditions, is used for HPLC and analyzes.Peace (Dionex) high performance liquid chromatograph is worn in employing, chromatographic condition: chromatographic column is a ShodexAsahipak NH2P-504E nh 2 column; Moving phase is acetonitrile: 0.01M ammonium acetate=40:13 (volume ratio); Flow velocity, 0.5mL/min; Detector, the RI-101 differential refraction detector; Column temperature, 30 ℃.
Embodiment 3
This description of test utilizes the resting cell of Pseudomonas putida GNA5 bacterial strain to prepare the method for D-glucosamine acid.
Fermention medium is formed (g/L): glucose 7, urea 7, corn steep liquor 2 (v/L), potassium primary phosphate 2, peptone 5, bitter salt 0.5, pH=7.0.Pseudomonas putida (Pseudomonasputida) GNA5 bacterial strain is inoculated in fermention medium with the inoculum size of 10% (percent by volume), culture temperature is 30 ℃, 180 rev/mins of shaking speed, when fermentation time reaches 8 hours, under aseptic condition, add aseptic D-glucosamine hydrochloride/D-glucosamine vitriol in nutrient solution, making its final concentration is 25mM/L.When fermentation time is 24 hours, stop fermentation.At 8000 rev/mins, centrifugal 15min under 4 ℃ of conditions obtains preliminary resting cell with fermented liquid.Is 50mM with this resting cell with 4 ℃ concentration, and the phosphoric acid buffer of pH7.0 is centrifugal with same condition once more after suspending, and repeats twice, can obtain to transform the resting cell of usefulness.
In resting cell, add bio-transformation substrate (being equivalent to 0.045g stem cell/g D-glucosamine hydrochloride) according to 0.1g cell/mM substrate, 30 ℃ of following oscillatory reactions 10 hours, generate D-glucosamine acid, transformation efficiency reaches 90%.
Embodiment 4
This description of test utilizes the purifying and the structure verification of the product of Pseudomonas putida GNA5 bacterial strain preparation.
Fermented liquid at 6000 rev/mins, behind the centrifugal 15min, is obtained supernatant liquor under 4 ℃ of conditions; Add isopyknic ethanol in this supernatant liquor, place in 4 ℃ of refrigerators and spend the night, suction filtration gets white crystals shape material.This white crystals shape material is water-soluble, and ethanol sedimentation obtains highly purified product once more.Highly purified product being carried out nuclear magnetic resonance spectrum (1H-NMR, 13C-NMR) analyze, is reference with D-glucosamine acid standard substance (purity〉97%) nuclear magnetic resonance spectrum.
It is in full accord that the NMR (Nuclear Magnetic Resonance) spectrum figure of this institute system crystallized product and Tokyo change into the D-glucosamine acid standard substance collection of illustrative plates of 97% purity that Co., Ltd. provides, and shows that Pseudomonas putida GNA5 oxidation D-glucosamine products therefrom is D-glucosamine acid.
Claims (9)
1. one kind transforms the bacterial strain that D-glucosamine prepares D-glucosamine acid, it is characterized in that this bacterial strain is a pseudomonas putida GNA5 bacterial strain, by China's typical culture collection center preservation, preserving number: CCTCC NO:M 208138.
2. one kind prepares the method for D-glucosamine acid with the described bacterial strain of claim 1, it is characterized in that placing the substratum oxidative fermentation that contains D-glucosamine directly to accumulate D-glucosamine acid described pseudomonas putida GNA5 bacterial strain, described D-glucosamine adds with the form of D-glucosamine hydrochloride or D-glucosamine vitriol, and D-glucosamine hydrochloride in the described substratum or D-glucosamine sulfate concentration are smaller or equal to 0.4mol/L.
3. the method for preparing D-glucosamine acid according to claim 2, the content that it is characterized in that carbon nitrogen source in the described substratum is smaller or equal to 25g/L.
4. the method for preparing D-glucosamine acid according to claim 3 is characterized in that described carbon source is a glucide.
5. the method for preparing D-glucosamine acid according to claim 4 is characterized in that described sugar is glucose.
6. the method for preparing D-glucosamine acid according to claim 2 is characterized in that described D-glucosamine hydrochloride or D-glucosamine vitriol are mixed with the aqueous solution earlier, regulates pH to 6.5-7.5 with NaOH again, is added into substratum then.
7. one kind prepares the method for D-glucosamine acid with the described bacterial strain of claim 1, comprising:
(1) with the described pseudomonas putida GNA5 of claim 1 bacterial strain, in substratum, carry out routine and cultivate, obtain resting cell;
(2) D-glucosamine is mixed with solution, as the bio-transformation substrate, this concentration of substrate is smaller or equal to 0.7mol/L;
(3) carry out conversion reaction in the substrate solution with (1) gained resting cell adding (2), generate D-glucosamine acid, the add-on of described resting cell is the 0.02-0.2g/mM substrate, and the conversion reaction temperature is 25-40 ℃, and the conversion reaction time is 1-48 hour.
8. the method for preparing D-glucosamine acid according to claim 7 is characterized in that the substratum that pseudomonas putida GNA5 strain fermentation is used in the step (1) is carbon source with glucose.
9. the method for preparing D-glucosamine acid according to claim 7 is characterized in that the D-glucosamine in the described bio-transformation substrate adds with the form of D-glucosamine hydrochloride or D-glucosamine vitriol.
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